Catalytic enzyme-modified textiles for active protection from toxins

ABSTRACT

Catalytic enzyme-modified textiles are disclosed for providing protection from chemical exposure. The textiles are composed of a cloth substrate, at least one polyelectrolyte layer, at least one enzyme layer to degrade the chemical agent, and at least one capping layer. Also disclosed is the related method for making catalytic enzyme-modified textiles.

This is a continuation-in-part application of application Ser. No.10/750,637 filed on Dec. 23, 2003, issued on Jun. 27, 2006 as U.S. Pat.No. 7,067,294, the entire contents of which are incorporated herein byreference.

BACKGROUND

1. Field of the Invention

The present invention relates to catalytic enzyme-modified textiles,and, more specifically, to catalytic enzyme-modified textiles for activeprotection from air or water borne toxins by active passivation andadsorption of toxic materials. Toxins include chemical and biologicalagents and toxic industrial chemicals.

2. Description of the Prior Art

There is an urgent need for the development of effective means toprotect people and the environment from the exposures of toxic chemicalsand other threat agents irrespective of the cause of exposure,accidental or due to terrorist act. Moreover, there is a need to protectpeople from exposure to chemicals during their work and from prolongedexposure to small amounts of toxic chemicals (especially in a closedenvironment). Long-term exposure to chemicals at low levels orpersistent encounters with small quantities of toxic chemicals may bemore harmful than one time exposure at higher levels. Examples of suchchemicals are pesticide and chemical warfare agents, and toxic vaporsfrom hydrolyzed chemical agents (e.g., HF and HCN).

The existing technologies use barrier protection involving materials ofhigh absorbing capacity to protect people and the environment. The mostwidely used adsorbent is active charcoal, which leads to the developmentof bulky materials. Materials used in barrier protection are bulky andhave only one useful life cycle. While the barrier technologies provideadequate protection, they have the serious technical problem of offgassing and disposal of the materials at the end of their active lifecycle because of the presence of toxic materials in concentrated form.Other concerns include weight, capacity, and inconvenience duringpractical use.

Many existing protective garments are heavy, bulky, and uncomfortable.They are usually made from rubber and other polymers. These garmentsgenerally provide passive, rather than active protection. That is, theyact simply as barrier layers to prevent contact of the chemical with theperson's body. Because they do not self-decontaminate after exposure toa chemical toxin, current protective garments require cleaning after usebefore they can be used again or before disposal in the case of singleuse garments.

Another existing technology regarding toxic chemicals is the use ofenzymes. Enzymes are the most effective catalyst against chemical agentsbut have limited long-term stability. Also, they often lose theircatalytic activity during immobilization steps. See G. F. Drevon, K.Danielmeier, W. Federspiel, D. B. Stolz, D. A. Wicks, P. C. Yu, A. J.Russell, “High-Activity Enzyme-Polyurethane Coatings,” Biotechnology andBioengineering, 79 (7) 785-794 (2002); and G. F. Drevon & A. J. Russell,“Irreversible Immobilization of Diisopropylfluorophosphatase inPolyurethane Polymers,” Biomacromolecules, 1 (4) 571-576 (2000), theentire contents of both are incorporated herein by reference. Lack ofstability and loss of catalytic activity render enzymes unsuitable forprotection applications. Several techniques have been reported forstabilizing the enzymes—most of them focusing on their immobilization toa suitable substrate. However, chemical linking to the surface causesthe enzymes to lose their activity substantially. Non-covalentimmobilization of enzymes on vesicles provides an effective means toretain enzyme activity. See U.S. Pat. No. 5,663,387 to Singh, the entirecontents of which is incorporated herein by reference. Deposition of asingle layer of enzymes on a surface is good for a sensor application,but not adequate for chemical agent passivation applications, whichrequire a larger amount of enzymes to effectively hydrolyze the toxicchemicals.

SUMMARY

The aforementioned problems are overcome by the present inventionwherein bioactive catalytic enzyme-modified textiles for providingprotection from chemical exposure that are stable and retain theircatalytic activity comprise a cloth substrate, at least onepolyelectrolyte layer, at least one enzyme layer to degrade the chemicalagent, and an end-capping layer. The present invention provides novel,bioactive, textiles for providing protection against chemical agents,which are more effective than barrier protection. These textiles can beused to develop lighter weight clothing to adsorb and passivate toxinsbefore they reach the human body. The textiles of the present inventioncan be used for reusable clothing that decontaminates itself afterexposure to toxins and can be worn multiple times or for disposableclothing and wipes intended for a single use that decontaminatethemselves without harming the environment.

In a preferred embodiment, the present invention takes advantage ofsuperior catalytic activity of enzymes by immobilizing them withinpolyelectrolyte multilayers (PEMs). The technique for formingmultilayers is simple and effective as polyelectrolytes of opposingpolarity are alternatively deposited through neutralization andovercompensation of their charges. See G. Decher, “Fuzzy Nanoassemblies:Toward Layered Polymeric Multicomposites,” Science, 277, 1232-1237(1997), the entire contents of which is incorporated herein byreference. Enzymes immobilized in the multilayers are easily accessibleto the incoming toxic materials and, thus, passivate them efficiently. Acapping agent provides stability to the multilayers, keeps enzymesprotected in adverse working environments, and attracts the toxic agentsto facilitate contact with the catalytic sites.

The present invention provides several advantages over the prior art. Itleads to enhanced enzyme shelf life under normal storage conditions. Itallows incorporation of multiple components into multilayers to provideadd-on capabilities to the packaged system. It is lightweight, robust,sturdy, disposable, self-decontaminating, and cost-effective. It offersversatility as it can be designed for use on various materials.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features, and advantages of the invention, aswell as the invention itself, will become better understood by referenceto the following detailed description, appended claims, and accompanyingdrawing where:

FIG. 1 shows an OPH−PEM preparation scheme. The completion of step 6marks the end of each OPH deposition cycle. [BPEI] and [PSS] quoted arecalculated based on monomer units of each polymer.

DETAILED DESCRIPTION

The core of the present invention is the packaging of essentialcomponents within alternate layers, or within a single layer, to producebioactive textiles and the stabilization of catalytic components andmultilayer assemblies to make them durable without losing theirperformance. Catalysts are immobilized within polyelectrolytes todegrade chemical agents and selectively capture degradation products. Acapping layer provides structural robustness and resists aggressivephysical and chemical perturbations.

In a preferred embodiment, the catalysts include enzymes, classlessnon-specific catalysts, and adsorbent particles. Preferred enzymes arethose that are superior catalysts for degrading chemical agents withhigh turnover numbers. Based on the need and application, anycommercially available enzyme can be used. Examples of preferred enzymesinclude organophosphorous hydrolase (OPH) (EC 3.1.8.1),organophosphorous acid anhydrolase (OPAA) (EC 3.1.8.2),diisopropylfluorophosphatase (DFPase) (EC 3.1.8.2), phosphotriesterases(PTE) (EC 3.1.8), and combinations of enzymes capable of passivating alarge number of toxic agents. A combination of OPH or PTE with OPAA willdestroy most of the chemical agents used in warfare. OPH is preferreddue to its good activity, stability, and ease of use. Several otherenzymes can be used, including paraoxonase, aryldialkylphosphatase, andbacterial HD hydrolase.

Classless non-specific catalysts catalyze hydrolysis of chemical agentsat a slower rate than enzymes. Examples of preferred classlessnon-specific catalysts include metal chelated catalytic particles (MCCP)such as metal chelated polymers (e.g., N-substitutedethylenediamine-copper (EDA-Cu²⁺) complexes), silica particles, andtitania (TiO₂) particles. TiO₂ particles are useful for light induceddegradation of chemical and biological agents because they haveappropriate oxidizing or reducing power during UV illumination due totheir band gap so as to decompose target particles. TiO₂ bandgap can betuned to allow visible light excitation through particle doping (W.Zhao, W. Ma, C. Chen, J. Zhao, Z. Shuai, “Efficient Degradation of ToxicOrganic Pollutants with Ni₂O₃/TiO_(2-x)B_(x) under Visible Irradiation,”J. Am. Chem. Soc., 126, 4782-4783 (2004), the entire contents of whichis incorporated herein by reference). MCCPs are useful in degradingthose chemical agents that are not degraded by enzymes. Multilayers willprovide enough chemical protection and separation to TiO₂ and enzymesthat they can function independently.

Adsorbent particles are functional catalytic particles (FCP) made byincorporating quaternary ammonium surfactant to silica microparticles.Also, acidic or basic alumina may be used to capture degradationproducts and biological particles. FCPs partially hydrolyze chemicalagents and selectively capture degradation products.

Multilayers can be fabricated on any sort of material, comprised ofeither natural or man-made polymer or glass, which can adsorb thecharged polymer components of the multilayers. Preferred examples ofmaterials include fiberglass and cotton. Man-made substances, such asrayon, nylon, etc., can also be used. Cotton may be used unmodified ormodified with cyclodextrin or an amine. The material can be used in manyforms, including cloth, thread, string, string knitted to cloth, etc.

Multilayers can also be fabricated on materials that are normallythought of as inert, provided that their surface is first chemicallymodified to generate functional groups that can support subsequentadsorption of the charged polymers or enzymes. For example, films ofpolytetrafluoroethylene (PTFE) or Teflon® are normally non-adhesivematerials; i.e, it is difficult to adsorb other materials to thesefilms. However, brief oxidation of the surface using a plasma createshighly acidic surface hydroxyl groups, which readily deprotonate to formanionic functional groups on the surface. Aminosiloxane self-assembledmonolayers can be chemisorbed to these species, providing a reactiveamine terminated monolayer coating on the PTFE or Teflon® film. Thesereactive amines can be used to bind other materials, such as metals,with good adhesion to the underlying PTFE or Teflon® (T. G. Vargo, J. A.Gardella Jr., J. M. Calvert, M-S. Chen, “Adhesive ElectrolessMetallization of Fluoropolymeric Substrates,” Science, 262, 1711-1712(1993), the entire contents of which are incorporated herein byreference). Because the surface hydroxyl groups present on plasmaoxidized films of PTFE or Teflon® deprotonate in water to form surfaceanions, adsorption of a cationic polyelectrolyte such as the branchedpolyethylenimine (BPEI), which is used as the first layer of themultilayer films described below, on these surfaces is possible. Inaddition, the possibility also exists to use a PTFE or Teflon® filmbearing a chemisorbed aminosiloxane film as a base positively chargedfilm in the multilayer assemblies described below. Such an aminosiloxanefilm might be used to directly bind an enzyme layer during thefabrication of a multilayer film. Alternatively, an aminosiloxane filmmight be used to bind a layer of anionic polyelectrolytes, such aspolystyrenesulfonate (PSS), which could then serve as a base layer forbinding the first layer of BPEI.

A second example of a material that is generally inert towards chemicalreactivity is diamond. However, through gentle plasma oxidation, surfaceOH groups can also be created on the diamond surface. Once again, suchgroups can serve to anchor other materials, such as aminosiloxane films(M-S. Chen, C. S. Dulcey, S. L. Brandow, D. N. Leonard, W. J. Dressick,J. M. Calvert, C. W. Sims, “Patterned Metallization of Diamond andAlumina Substrates,” J. Electrochem. Soc., 147 (7) 2607-2610 (2000), theentire contents of which is incorporated herein by reference), thatserve to impart chemical reactivity or binding abilities to the diamondsubstrate.

A molecular “glue” is used to hold all the active catalytic componentstogether, to stabilize enzymes, and to provide adequate adhesion of theassemblies to the support materials without involving any chemicalreaction. Polyelectrolytes, by virtue of available cationic or anionicfunctionalities in abundance, provide an excellent means to glue themolecular components. Cooperativity and electrostatic interactions,hydrogen bonding, and/or Van der Waals interaction between anionic andcationic sites leads to the formation of strong association ofmultilayers. Examples of polyelectrolytes that can be used includecommercially available polyelectrolytes, branched or linearpolyethyleneimine (PEI), polyacrylic acid (PAA), polymethacrylic acid(PMA), polystyrene sulfonate (PSS), polydiallyl dimethyl ammoniumchloride (PDDA), polyvinylpyridine (PVP), polyvinyl sulfate (PVS),polyallylamine hydrochloride (PAH), their chemically alteredderivatives, and any combination thereof.

The surface is chemically tuned through composition of thepolyelectrolyte deposition solution. For example, in neutral or basicsolution where the pH is greater than the isoelectric point of silica orglass, the silica or glass surface is negatively charged and readilyadsorbs cationic polyelectrolytes like BPEI. However, if one needed towork in highly acidic media due to stability or solubilityconsiderations for the polyelectrolyte to be deposited, one could modifythe surface with an aminosiloxane film to create a positively chargedsurface. One could then bind a polyelectrolyte such as polystyrenesulfonate (PSS), which remains an anionic species even in highly acidicsolution (e.g., pH ˜1), to recreate a negatively charged surfacesuitable for deposition of further polyelectrolyte layers at low pH.

The deposition method may be variable. For example, a simple dip coatingprocedure involving immersion of the substrate to be treated in theappropriate polyelectrolyte of enzyme solutions provided the bestperforming cloths with respect to catalytic methyl parathion (MPT)hydrolysis to paranitrophenol (PNP) in solution. However, spray coatingor spin coating methods, which are readily amenable for commercialapplications, can also be used to fabricate the catalytic multilayers onthe substrates. These latter methods produced multilayer filmsexhibiting less catalytic activity than samples prepared by dip coating.However, since no attempt was made to optimize multilayer depositionaccording to spin coating or spray coating methods, further improvementin catalytic ability should be possible for such films.

A capping agent is used to encase the catalytic components. The cappingagent provides stability to the catalytic components, keeps the enzymearchitecture dimensionally protected in adverse working environments,and ideally attracts the toxic agents to facilitate contact with thecatalytic sites. In a preferred embodiment, pH- and photo-polymerizablemonomers and/or metal-ion crosslinked systems are used as cappingagents. In an even more preferred embodiment, the capping agent isselected from the group consisting of 1,2-dihydroxypropyl methacrylate(DHPM), 1,2-dihydroxypropyl 4-vinylbenzyl ether (DHPVB), andN-[3-(trimethoxysilyl)propyl]ethylenediamine (TMSED). Preferably,polyamine silane derivatives, in addition to capping agentcross-linkable polyelectrolytes, can be used. Polyelectrolyte cappinglayers can also be varied to include BPEI and other amine-bearingpolyelectrolytes, such as N-[3-(trimethoxysilyl)-propyl]ethylenediamine(TMSED, hydrolyzed and crosslinked via siloxane bond formation afterdeposition).

Aqueous, pH 8.6 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP)buffer is a preferred rinsing agent and component of BPEI solution usedto coat a previously deposited layer of OPH enzyme. Note that use of awater rinse after deposition of an OPH layer followed by attempteddeposition of a BPEI layer using an aqueous BPEI solution not containingBTP buffer, leads to extraction of a portion of the previously depositedOPH enzyme from the substrate and results in poor quality multilayerfilms having low catalytic activities. The reason for this loss of OPHenzyme from the surface is not well understood. However, use of the BTPbuffer during the rinse and as a component in the BPEI solution duringdeposition of the BPEI layer capping the OPH enzyme layer minimizes OPHextraction from the surface. Apparently the BTP interacts with theimmobilized OPH enzyme to increase its adhesion to the underlying BPEIlayer in the film. The same phenomenon can be applied to other enzymes,but with differing intensity in BTP enzyme affinity.

In contrast, attempts to deposit an OPH enzyme layer onto a BPEI layerthat had been deposited from a BPEI solution containing BTP buffer andrinsed with pure BTP buffer does not lead to deposition of large amountsof OPH reproducibly. OPH is most reproducibly deposited onto a BPEIlayer that has been deposited from an aqueous solution of BPEI notcontaining BTP buffer. Once again, the reason for this is not wellunderstood. Therefore, to deposit a multilayer film bearing more thanone OPH enzyme layer, one cannot use the capping layer of BPEI,deposited from BPEI solution containing BTP buffer, over the previousOPH as a base layer for direct deposition of the next OPH layer.Instead, one can first apply an intervening PSS layer on top of the BPEIlayer capping the previous layer of OPH. One can then use the PSS layeras a base layer to apply another BPEI layer using an aqueous BPEIsolution not containing the BTP buffer. In this manner, another layer ofOPH enzyme may then be readily and reproducibly added to the growingmultilayer film. Consequently, one such preferred processing scheme forthe successful fabrication of polyelectrolyte-OPH enzyme multilayerfilms is shown in FIG. 1.

Processing is versatile. Woven textiles and fabrics may be used directlyas substrates for deposition of the catalytic multilayer films.Alternatively, the multilayers may be deposited onto threads, which maybe subsequently woven into fabrics of the desired shape. The enzymecapping layers provide sufficient protection to the underlying enzymelayer(s) such that the degree of abrasion and wear that the multilayercoated thread experiences during the weaving process is not sufficientto eliminate the catalytic activity of the resulting woven cloth.

Optionally, the top layer can be designed to kill bacteria and viruses.For example, by modifying the top layer BPEI amine with a hexyl groupand quaternizing with methyl bromide, the top layer will be abactericidal layer. See J. Lin, S. Qiu, K. Lewis, A. M. Klibanov,“Bactericidal Properties of Flat Surfaces and Nanoparticles Derivatizedwith Alkylated Polyethylenimines,” Biotechnol. Prog., 18, 1082-1086(2002), the entire contents of which are incorporated herein byreference.

Having described the invention, the following examples are presented toillustrate specific applications of the invention, including the bestmode currently known to perform the invention. It is understood thatthese specific examples are not intended to limit the scope of theinvention described in this application.

EXAMPLES

General Considerations

Materials: All chemicals used were A.C.S. reagent grade or better fromSigma-Aldrich Chemical Co. and were used as received unless otherwisenoted. Deionized water (18 MΩ-cm resistivity) was used for allexperiments. Nitrogen gas for drying samples from liquid N₂ boil-off waspassed through a 0.2 μm PTFE filter prior to use. β-cyclodextrin wasobtained from Cargill Cerestar Inc. Branched polyethylenimine (BPEI)(CAS No. 25987-06-8) was a 50% wt. solution in water (Aldrich ChemicalCo., catalog no. 181978, batch no. 16104HA) with M_(n) (GPC) ˜60000g/mole and M_(w) (LS) ˜75000 g/mole. Poly(sodium 4-styrensulfonate)(PSS) (Aldrich Chemical Co., catalog no. 24,305-1, Lot no. 20025CU, CASNo. 25704-18-1) had M_(w)˜70000 g/mole. Organophosphorous hydrolase(OPH) (EC 3.1.8.1) enzyme was received as freeze dried powder fromAberdeen Proving Grounds, MD, and stored in the refrigerator at 4° C.until needed for experiments. The amount of OPH enzyme was determined byestimating the total protein content and performing an enzyme assay onthe samples (Y. Lee, I. Stanish, V. Rastogi, T. Cheng, and A. Singh,“Sustained Enzyme Activity of Organophosphorus Hydrolase in PolymerEncased Multilayer Assemblies,” Langmuir, 19, 1330-1336 (2003), theentire contents of which are incorporated herein by reference). OPHenzyme samples used in our studies were technical grade (5% enzyme mixedwith ˜95% of trehalose along with small amounts of non-enzymaticprotein). Lint-free clean room paper towels (Model 8025 Clean RoomWipes) were obtained from Liberty Industries, Inc. A clean 100% cottonundershirt (Hanes Inc.) provided cotton cloth samples. Cotton thread was100% cotton Mouliné Spécial DMC 25 from DMC S. A., F5579 Paris cedex 12,France. Fiberglass woven cloth was style #106 from Hexcel Schwebel(piece #6493106201; sample #3664). This material had a plain weavestyle, a wrap count of 56, a fill count of 56, a fabric thickness of0.04 mm, and a breaking strength of 45 lb·f/in.

Solutions: Stock aqueous solutions were prepared as described below.Polymer and buffer solutions were stable for at least two weeksfollowing preparation. OPH enzyme solutions and CoCl₂ cofactor solutionswere prepared fresh daily as needed. The stock solutions included:

1. BTP: This buffer was prepared as a 0.01 M solution by dissolving2.823 g Bis-tris propane (i.e.,1,3-bis[tris(hydroxymethyl)methylamino]propane) in ˜950 mL water in a 1L volumetric flask. A 0.1 M HCl(aq) solution was then added dropwise tobring the BTP solution to pH 8.6, followed by dilution with water to 1 Lin the volumetric flask and mixing.

2. CHES-w: This pH 8.6 buffer solution was prepared as a 0.01 M solutionby dissolving 2.07 grams of 2-[N-Cyclohexylamino]ethanesulfonic acid ina 1 L volumetric flask containing ˜900 mL water. A sufficient volume of1 M NaOH(aq) solution was added dropwise to adjust the solution pH to8.6 followed by the addition of water to make up volume to 1000 mL.

3. CHES-m: CHES-m buffer (0.008 M, in 20% methanol) was prepared bypipetting 200 mL methanol into a 1 L volumetric flask and mixing it withCHES-w buffer to make 1 L volume.

4. BPEI-w: A 1.5 g sample of BPEI was added to a 200 mL portion ofdeionized water with magnetic stirring and diluted to make a 1 Lsolution. The pH of the solution was adjusted to 8.6 by addition ofsmall volumes of 2% HCl solution in water.

5. BPEI-b: This polymer solution was prepared by placing 600 mg of BPEIsolution into a 250 mL beaker and adding 100 mL BTP buffer with magneticstirring. The volume was then made up to 500 mL by adding additionalvolume of BTP buffer. The pH of the solution was 8.6.

6. PSS-w: A 1.03 g portion of PSS was dissolved in 1 L water to make a0.005 M solution. This solution resulted in a pH of 6.6.

7. Stock MPT: A 26.2 mg (100 μmole) portion of methyl parathion (MPT)(MW=263.22 g/mole) was placed in a 1 L volumetric flask and 200 mL ofmethanol was added by pipet. CHES-w buffer was slowly added in ˜50 mLaliquots with thorough mixing until the total volume of solution was 1L. The solution was mixed and transferred to polyethylene containers,which were stored in the refrigerator at 4° C. to inhibit decompositionuntil needed for experiments. The MPT concentration of this solution was100 μM, and the solution had pH ˜8.6.

8. Stock PNP: A 13.9 mg (100 μmole) portion of p-nitrophenol (PNP)(MW=139.1 g/mole) was placed in a 1 L volumetric flask and 200 mL ofmethanol was added by pipet. CHES-w buffer was slowly added in ˜50 mLaliquots with thorough mixing until the total volume of solution was 1L. The solutions was mixed and transferred to polyethylene containers,which were stored in the refrigerator at 4° C. until needed forexperiments. The PNP concentration of this solution was 100 μM and thesolution had pH ˜8.6.

9. Stock Co: This co-factor solution was prepared by dissolving ˜1.2 mg(5 μmole) CoCl₂·6H₂O (MW=237.93 g/mole) in ˜40 mL BTP in a 50 mLvolumetric flask, diluting to the 50 mL mark with BTP, and mixing. Thesolution contains ˜100 μM Co(II) in ˜0.01 M BTP at pH ˜8.6. Thissolution is prepared fresh as needed.

10. Stock OPH: This enzyme solution is prepared by dissolving ˜4 mg OPHenzyme in 16 mL of freshly prepared Stock Co solution. This solution wasprepared fresh each time. This solution was placed in the refrigerator(4° C.) between each deposition cycle to avoid enzyme inactivation.

Substrate Preparation and Cleaning: Cotton cloth samples were cut fromthe undershirt and cotton string samples were cut from the thread usingscissors as needed. Both the cotton cloth and cotton thread were useddirectly in experiments without further cleaning. Fiberglass cloth wascut into a size appropriate for each experiment with scissors and theedges were fused by melting in a propane-air flame to prevent unravelingof the weave during subsequent handling. Fiberglass cloth samples werecleaned by a two-step process, hereafter referred to as the StandardCleaning Protocol, prior to use. The fiberglass cloth was first immersedin a solution comprising a 1:1 v/v mixture of concentrated hydrochloricacid in methanol in a fume hood for ˜1 hour. The sample was stirred witha glass-stirring rod periodically to dislodge any gas bubbles trapped onthe weave. The fiberglass cloth was then rinsed thoroughly by immersionin separate containers of water at least 4 times. It was next immersedin concentrated sulfuric acid for ˜1 hour. A clean glass-stirring rodwas again used to dislodge any bubbles trapped in the weave. (Fiberglasscloth samples could be readily stored for at least 4 days in thiscleaning bath without contamination.) The fiberglass cloth sample wasthen removed from the sulfuric acid and washed thoroughly with water asdescribed for the HCl/methanol treatment. The sample was removed fromthe final water rinse and placed on a lint-free clean room paper towel(Model 8025 Clean Room Wiper; Liberty Industries, Inc.). The sample wasstretched out flat on the towel by hand, covered with a second lint-freetowel, and pressed to adsorb most of the water from the fiberglasscloth. Powder-free latex gloves were worn during this process to preventcontamination of the fiberglass cloth by oils from the bare hand.Residual moisture was removed from the fiberglass cloth using the streamof N₂ gas filtered through a 0.22 μm PTFE filter. The dried, cleanfiberglass cloths were used immediately for coating experiments asdescribed in subsequent examples to avoid contamination. Cleanfiberglass cloth samples for use in experiments scheduled later in thesame day were stored immersed in the final water rinse until needed.

General Method for Multilayer Fabrication: This section outlines thegeneral protocol for deposition of the polyelectrolyte and enzymemultilayers onto cloth substrates. In general, polyelectrolytemultilayers containing encapsulated enzymes were prepared by sequentialtreatment of fiberglass cloth or cotton cloth substrates withappropriate polyelectrolyte solutions and enzyme solutions usingdipping, spraying, or spin coating methods. The enzyme layers wereapplied by replacement of anionic polyelectrolyte layers by enzymesduring the deposition process. Therefore, in general, the substrate wasfirst coated with a cationic polyelectrolyte layer. Thereafter,alternating layers of negatively charged and positively chargedpolyelectrolytes were coated onto the first cationic polyelectrolytelayer. The enzyme was immobilized by replacement of anionicpolyelectrolyte solution by the enzyme solution during the depositionprocess. Following deposition of each polyelectrolyte and enzyme layer,the substrate was washed using water or BTP buffer to remove looselyadsorbed material prior to deposition of the next layer of themultilayer film. In all cases, each enzyme layer was deposited onto andcapped by an adsorbed layer of cationic polyelectrolytes to encapsulatethe enzyme in the multilayer film. After completion of the multilayerfabrication process, samples were subjected to a Standard DryingProtocol involving placement in a lyophilizer and drying under vacuum(1.3-1.5 μbar) for 2 hours. Samples were then stored in a refrigerator(4° C.) until needed for experiments. In general, samples were used theday after preparation unless noted otherwise. Specific treatmentsequences are described in the examples that follow. FIG. 1 shows oneexample of a protocol for fabrication of the film with specificconditions noted.

Standard Enzyme Activity Assay: The activity of the encapsulated enzymesin the multilayer films was tested via hydrolysis of methyl parathion(MPT) to paranitrophenol (PNP). An extinction coefficient of 9300L·mole⁻¹ cm⁻¹ at 275 nm was first determined for MPT by appropriatedilution of the Stock MPT solution with CHES-m buffer. A series ofdilutions of the Stock MPT solution with CHES-m buffer was thenperformed and the absorbance of each solution was measured at 275 nm ina 1.00 cm pathlength cell. A linear calibration curve of absorbance,A₂₇₅, vs. [MPT] (in μM) was then constructed. The calibration curve wasdescribed by equation (1):A ₂₇₅=0.0093·[MPT] r ²=0.9999  (1)

The analysis of PNP was performed in a more strongly basic solution tomaximize the concentration of the strongly absorbing paranitrophenolateanion species. A series of dilutions of the Stock PNP solution were madeusing the CHES-m buffer to prepare a calibration curve. A 600 μL aliquotof each diluted PNP solution was separately mixed with a 900 μL aliquotof freshly prepared 1 mM NaOH(aq) solution (i.e., dilution factor=2.5)and the absorbance was measured at 405 nm in a 1.00 cm pathlength cell.An extinction coefficient of 14,000 L·mole⁻¹ cm⁻¹ was measured for thePNP anion. A linear calibration curve, corrected for the 2.5 dilutionfactor, described by equation (2) was obtained:A ₄₀₅=0.0140·[PNP] r ²=0.9998  (2)

The analysis of the activity of the encapsulated enzymes in themultilayer films was carried out by immersing the substrate coated bythe polyelectrolyte-enzyme multilayer film in a beaker containing 100 mLof stirred Stock MPT solution. At various times afterwards, 600 μLaliquots of the MPT solution were withdrawn, diluted with 900 μL offreshly prepared 1 mM NaOH(aq) solution, and mixed. The absorbance ofthe paranitrophenolate anion was measured at 405 nm and the [PNP] wasdetermined from the calibration curve. Plots of [PNP] vs. immersion timewere constructed to map the activity of the polyelectrolyte-enzymemultilayer film cloth samples.

Standard Enzyme Binding Assay: Protein assays were carried out todetermine the quantity of OPH bound in multilayer assemblies on variousfabric substrates described in subsequent examples according to theliterature method (Y. Lee, I. Stanish, V. Rastogi, T. Cheng, A. Singh,“Sustained Enzyme Activity of Organophosphorus Hydrolase in PolymerEncased Multilayer Assemblies,” Langmuir, 19, 1330-1336 (2003), theentire contents of which is incorporated herein by reference). Briefly,a sample (˜100 mg) of fabric coated with an OPH-polyelectrolytemultilayer assembly was digested with agitation for ˜2 hours in 1.5 mLof a 2 M NaCl(aq) solution and then centrifuged at 5000 rpm for 3minutes. Aliquots of Biuret reagent (2.2 mL) and Folin and Ciocalteu'sphenol reagent (0.1 mL) were added to a 0.2 mL aliquot of thesupernatant isolated during centrifugation and the absorbance of theresulting solution at 720 nm was measured. Protein content wascalculated by comparison to standard curves prepared using knownconcentrations of OPH. Hereafter, this method will be referred to as theStandard Enzyme Binding Assay.

Example 1 Fabrication of Polyelectrolyte-OPH Enzyme Multilayer onFiberglass Cloth by Dip Coating

This example demonstrates the preparation of a polyelectrolyte-enzymemultilayer containing a single layer of the OPH enzyme on a wovenfiberglass cloth sample by the dip coating method.

Two circular samples of fiberglass cloth of ˜3 inch diameter each werecut from the roll of cloth obtained from the supplier (Hexcel) andcleaned using the Standard Cleaning Protocol. Separate plasticcylindrical 50 mL centrifuge tubes (Falcon BD), each of length ˜4.5inches, were used to secure the samples. The centrifuge tube diameterwas constant at ˜1.25 inches over a 4 inch length of the tube, rapidlytapering to ˜0.25 inches at the bottom of the tube. Each tube wasprepared for use by drilling a hole through the 0.25 inch diameterplastic section of the bottom of the tube to equalize air pressure andpermit free flow of solution into and out of the tube during sampletreatment. The freshly cleaned fiberglass cloths were stretched tightlyby hand over the threaded mouths of the centrifuge tubes and securedwith rubber bands. Excess fiberglass cloth was trimmed away usingscissors or a razor blade until flush with the rubber band anddiscarded. Each centrifuge tube, affixed with the sample cloth, wasmounted vertically with the vent hole facing upwards using a standardlaboratory clamp. A 250 mL beaker containing a Teflon® stirbar wasplaced on a magnetic stirrer immediately under each mounted sample. Analiquot of BPEI-w solution (˜100 mL) was added to the beaker andstirring was begun. Each centrifuge tube bearing the sample cloth wasthen lowered into the stirred solution until all portions of thefiberglass cloth were immersed and left for 10 minutes. The tubes werethen removed from the BPEI-w solution, unclamped, and gently shaken toremove adherent BPEI-w solution. A beaker containing 100 mL of stirredwater was placed on the stirring plate and the tubes were returned tothe clamp and lowered into the rinse water until each fiberglass clothwas again completely immersed. After 1 minute the tubes were removedfrom the rinse water, unclamped, and again gently shaken to removeexcess solvent. The sample cloth on the first tube was designated as acontrol cloth. It was dried further using a stream of filtered nitrogengas, subjected to the Standard Drying Protocol, and stored in therefrigerator until needed for further experiments.

After removal from the aqueous rinse solution, the tube bearing thesecond cloth was again clamped and immersed in a beaker containingstirred Stock OPH solution, which had been allowed to warm to roomtemperature after removal from storage in the refrigerator. Care wastaken to ensure that the entire cloth sample was immersed. After 10minutes, the tube was withdrawn from the OPH solution, unclamped, andagain shaken to remove excess solution. The tube was remounted in theclamp and the sample cloth immersed in a stirred BTP buffer solution for2 minutes. Following the rinse with BTP buffer, the tube was unclamped,shaken to remove excess buffer from the sample cloth, and remounted inthe clamp. The tube was then lowered into a stirred BPEI-b solution todeposit a capping layer of the BPEI polymer over the OPH enzyme layer onthe immersed sample cloth. After 10 minutes, the tube was removed fromthe BPEI-b solution, unclamped, and shaken to remove excess BPEI-bsolution. The tube was then returned to the clamp and the sample clothimmersed in stirred BTP solution for 1 minute as a final washing cycle.The tube was removed from the BTP buffer wash solution, unclamped, andgently shaken to remove excess BTP buffer solution. The sample cloth wasblown dry using a filtered nitrogen gas stream and separated from thetube by cutting the rubber band. The treated sample cloth wastransferred to the lyophilizer for drying using the Standard DryingProtocol. Dried sample cloths were then stored in the refrigerator untilneeded for experiments.

The multilayer film structure of the treated sample cloth isconveniently represented by the notation, FG/BPEI/OPH/BPEI, where FGrepresents the underlying fiberglass cloth substrate coatingsequentially by layers of BPEI polyelectrolyte, OPH enzyme, and acapping layer of BPEI polyelectrolyte. The corresponding notation forthe control fiberglass cloth is FG/BPEI. After the multilayerfabrication process was completed a 500 μL aliquot of the Stock OPH usedin the deposition was analyzed to confirm enzyme activity using theStandard Enzyme Activity Assay. OPH activity of the used solution wasidentical within experimental error (±5%) to that of the fresh enzymesolution.

Example 2 MPT Hydrolysis Activity of Multilayer-coated Fiberglass ClothContaining a Single OPH Enzyme Layer Prepared by Dip Coating

This example demonstrates that fiberglass cloth dip coated with amultilayer polyelectrolyte film encapsulating a single layer of OPHenzyme is capable of hydrolyzing MPT in solution to Pnp.

The control FG/BPEI (weight 80 mg) and sample FG/BPEI/OPH/BPEI cloths(weight 82 mg) prepared in Example 1 were removed from the refrigeratorand allowed to warm to room temperature. Each cloth was then fullyimmersed in a separate beaker containing 100 mL of stirred Stock MPTsolution for 22 hours at room temperature (23±2° C.). The clear andcolorless MPT solution containing the FG/BPEI/OPH/BPEI sample clothdeveloped a clear yellow color with increasing time. Little, if any,color was observed in the MPT solution containing the FG/BPEI controlcloth. The sample and control cloths were then removed from the MPTsolutions, rinsed carefully, blown dry with the filter nitrogen gasstream, and returned to storage in the refrigerator. The Standard EnzymeActivity Assay was performed for each of the MPT solutions. The assayindicated that only ˜0.8 μM PNP was produced in the solution exposed tothe FG/BPEI control cloth, consistent with negligible MPT hydrolysis. Incontrast, ˜18 μM PNP was detected in the MPT solution treated using theFG/BPEI/OPH/BPEI sample cloth, demonstrating the retained activity ofthe encapsulated OPH enzyme towards MPT hydrolysis in this sample.

Example 3 Reproducibility of Fabrication and Hydrolytic Activity ofMultilayer-coated Fiberglass Cloth Containing a Single OPH Enzyme LayerPrepared by Dip Coating

This experiment illustrates the degree with which the multilayer-coatedfiberglass cloths having MPT hydrolysis activity can be fabricated bythe dip coating method.

The fabrication procedure described in Example 1 was repeated 3 timesusing fresh samples of fiberglass cloth in each case. Fresh solutionswere used to coat the samples with encapsulated OPH enzyme towardshydrolysis of MPT as described in example 2. Once again, the 3 FG/BPEIcontrol clothes produced only ˜0.8 μM PNP in each case, indicatingnegligible hydrolysis of the MPT solution. The separately preparedFG/BPEI/OPH/BPEI sample cloths produced ˜15 μM PNP, ˜16 μM PNP, and ˜15μM PNP in good agreement with the 18 μM PNP produced using thecorresponding sample from Example 2. The average activity exhibited bythe 4 sample cloths of Examples 2 and 3 is 16 μM PNP with a standarddeviation (σ) of ±1.2 μM PNP, suggesting a reproducibility (2σ) of ˜15%for the fabrication and hydrolysis processes.

Example 4 Fabrication of Multilayer-coated Fiberglass Cloth ContainingMultiple OPH Enzyme Layers by Dip Coating

This example describes the preparation of fiberglass cloths coated bymultilayer films bearing more than one layer of enzyme. The exampleshows the fabrication of a film containing 4 layers of the OPH enzyme.

A sample of fiberglass cloth bearing a single layer of OPH enzyme wasfabricated as described in Example 1 using fresh OPH solution. However,following binding of the capping BPEI-b layer and subsequent rinse inthe BTP solution, treatment was continued by immersion for 10 minutes ina stirred PSS-w solution. The sample was then gently shaken to removeexcess PSS solution, rinsed for 1 minute in stirred water, and gentlyshaken to remove excess rinse water. The treatment sequence of Example 1was repeated to apply the next BPEI-w layer, the second OPH layer, andthe next BPEI-b layer. Following application of a second PSS-w layer andan aqueous rinse, the treatment sequence of Example 1 was againperformed to apply the third BPEI-w, OPH, and BPEI-b layers. Anotherlayer of PSS-w was then applied and the sample rinsed in water asdescribed above. The sample was then subjected to a final treatmentsequence as described in Example 1 to apply the fourth layers of BPEI-w,OPH, and BPEI-b. The multilayer sequence of treated sample cloth isFG/(BPEI/OPH/BPEI/PSS)₃/BPEI/OPH/BPEI. In general, a multilayercontaining “x” layers of OPH can be prepared through repetition of thetreatment sequence of Example 1 “x” times, using a PSS layer as aseparation polyelectrolyte layer between each repetition of thetreatment sequence of Example 1.

Example 5 MPT Hydrolysis Activity of Multilayer-coated Fiberglass ClothContaining Multiple OPH Enzyme Layers Prepared by Dip Coating

This example demonstrates that fiberglass cloth coated with a multilayerpolyelectrolyte film encapsulating 4 layers of OPH enzyme is capable ofhydrolyzing MPT in solution to PNP.

The ability of the sample cloth fabricated in Example 4 having themultilayer film structure FG/(BPEI/OPH/BPEI/PSS)₃/BPEI/OPH/BPEI tohydrolyze MPT solution was tested using the procedure described inExample 2. A freshly prepared control cloth having the structure FG/BPEIwas also tested. The control cloth produced ˜0.8 μM PNP, in agreementwith our observations in Examples 2 and 3. The sample cloth containing 4layers of OPH enzyme produced ˜31 μM PNP, a value about twice thatobserved for the sample cloths containing a single layer of OPH enzymein Examples 2 and 3. Consequently, sample cloths containing multipleenzyme layers are capable of hydrolyzing MPT in solution and can do soat a greater rate than cloths bearing only a single OPH enzyme layer intheir multilayer coatings.

Example 6 Time Dependence of the MPT Hydrolysis Activity of MultilayerFiberglass Cloths Bearing a Single OPH Enzyme Layer Prepared by DipCoating

This example illustrates the activity of a sample cloth bearing a singleOPH enzyme layer towards the repetitive hydrolysis of MPT in solution asa function of time since fabrication.

The fiberglass sample cloth (weight ˜80 mg) fabricated in Example 1having the multilayer structure FG/BPEI/OPH/BPEI was used for this test.This sample cloth was used to sequentially hydrolyze fresh 100 mLaliquots of Stock MPT solution as described in Example 2 during a ˜17day time period. Four separate MPT solutions were hydrolyzed during the5 working days in each week. Following each hydrolysis experiment, thesample cloth was rinsed with water for ˜1 minute and blown dry with afiltered stream of nitrogen gas before being used for the nextexperiment. The Standard Enzyme Activity Assay was used to measure thequantity of PNP produced in each MPT solution immediately aftercompletion of each 22 hour experiment. On Fridays and during theweekends, no hydrolysis experiments were conducted and the sample clothremained in storage in the refrigerator. The activity assay data isillustrated as a function of the number of hydrolysis cycles and age ofthe sample cloth in Table 1.

TABLE 1 Reuse of Fiberglass Sample Cloth Bearing a Single OPH Layer forMPT Hydrolysis [PNP] 18 17 13 12 15 7 9 9 15 6 5 8 Produced (μM) SampleAge 0 1 2 3 7 8 9 10 14 15 16 17 (Days) Hydrolysis 1 2 3 4 5 6 7 8 9 1011 12 Cycle Number

In general, the data indicate that the activity of the sample clothtowards hydrolysis of MPT decreases with the age and use of the samplecloth, with the sample reaching a value of ˜25-35% of its initialhydrolysis activity after ˜17 days of use. Consequently, the samplecloth retains measurable activity for the hydrolysis of MPT for at leasta 17 day period of repeated use. Hydrolysis cycle number 5 and 9represent experiments begun on the Monday of the work week followingstorage of the sample in the refrigerator over the weekend. These dataindicate that samples temporarily regain a portion of their lostcatalytic activity towards solution phase hydrolysis of MPT upon storagefor ˜3 days in the refrigerator during the 17 day time period covered bythese experiments.

Example 7 Time Dependence of the MPT Hydrolysis Activity of MultilayerFiberglass Cloths Bearing 4 OPH Enzyme Layers Prepared by Dip Coating

This example illustrates the activity of a sample cloth bearing 4 OPHenzyme layers towards the repetitive hydrolysis of MPT in solution as afunction of time since fabrication.

The fiberglass sample cloth (weight ˜82 mg) fabricated in Example 4having the multilayer structure FG/(BPEI/OPH/BPEI/PSS)₃/BPEI/OPH/BPEIwas used for this test. This sample cloth was used to sequentiallyhydrolyze fresh 100 mL aliquots of Stock MPT solution as described inExample 2 during a ˜20 day time period. Separate, fresh MPT solutionswere hydrolyzed each day that an experiment was run. Following eachhydrolysis experiment, the sample cloth was rinsed with water for ˜1minute and blown dry with a filtered stream of nitrogen gas before usein the next experiment. The Standard Enzyme Activity Assay was used tomeasure the quantity of PNP produced in each MPT solution immediatelyafter completion of each 22-hour experiment. During the weekends, nohydrolysis experiments were conducted and the sample cloth remained instorage in the refrigerator. Representative activity assay data isillustrated as a function of the number of hydrolysis cycles of thesample cloth in Table 2.

TABLE 2 Reuse of Fiberglass Sample Cloth Bearing 4 OPH Layers for MPTHydrolysis [PNP] Produced (μM) 31 14 22 20 21 24 20 19 22 23 Sample Age(Days) 0 1 2 3 8 9 10 14 17 20 Hydrolysis Cycle 1 2 3 4 5 6 7 8 9 10Number

The fifth hydrolysis cycle was performed following a long holidayweekend in which the sample had been refrigerated for ˜5 days. Theeighth hydrolysis cycle was performed after a three-day refrigerationover a weekend. The sample was also stored in the refrigerator for ˜2days following each of the eighth and ninth hydrolysis cycles. Theresults in Table 2 clearly indicate that hydrolysis of MPT continues tooccur at measurable rates for the sample bearing 4 OPH enzyme layers forextended periods after fabrication of the sample.

Example 8 Fabrication of Multilayer-coated Fiberglass Cloth Bearing aSingle Layer of OPH Enzyme by Spray Coating

This example describes the fabrication of a multilayer coatingcontaining one layer of OPH enzyme on a fiberglass cloth using a spraycoating method.

A plastic drink cup (32 U.S. ounce capacity; ˜4 inch diameter×5 inchheight) having a rim at its mouth with a corresponding plastic lidcapable of being secured to said cup when said lid is snapped over saidrim was modified as a sample holder for this experiment. A circular holeof ˜2.5 inch diameter, concentric with the center of the circular lid,was cut from said lid. The bottom of the plastic cup was next cut away,leaving an approximately cylindrical structure of diameter ˜4 inches andheight ˜4 inches that was open at both ends. A ˜5 inch diameter piece offiberglass cloth was cleaned using the Standard Cleaning Process andstretched taut over the mouth of the plastic cup. The fiberglass clothwas secured to the cup by snapping the lid onto the mouth of the cupover the stretched, taut, fiberglass cloth. In this manner, a circulararea of fiberglass cloth ˜2.5 inches in diameter was exposed in the areaof the lid that had been cut away and was available for sprayingtreatments. Excess cloth protruding from the lid along the rim of thecup was trimmed back to the rim with scissors or a razor blade. Thetrimmings were discarded.

A cardboard box of dimensions 16 inch height×12 inch width×10 inch depthwas placed in the fume hood to act as a catch basin for aerosols duringspraying such that one of the 16 inch×12 inch sides faced theexperimenter and a 12 inch×10 inch side comprised the top of the box.The top of the box and the front face of the box were cut away anddiscarded. A second cup, identical to the first and not modified in anyway, was obtained to act as a solution catch basin during spraying. Thissecond cup was mounted in an iron ring clamp inside the cardboard box inthe fume hood such that the mouth of the cup was tilted ˜45° from thevertical and faced the experimenter. The assembly comprising thefiberglass cloth sample secured to the first (bottomless) cup by themodified lid was then nested in the second cup mounted in the iron ringclamp in the hood.

Solutions of BPEI-w, BTP, and BPEI-b were freshly prepared and directlyloaded into separate Nalgene® aerosol spray bottles (Aldrich ChemicalCo., catalog no. Z27, 925-0). Stock OPH solution was filtered using a0.2 μm PTFE filter attached to a 5 mL plastic Fortuna® syringe toeliminate any insoluble particulates that could clog the tiny outlet ofthe spray bottle atomizer during the spraying process prior to loadinginto another spray bottle. Each solution was prepared for spraying bypressurizing the appropriate spray bottle using a detachable hand pump.The pump was depressed 20 times to charge the sprayer. The solution wasthen sprayed onto the fiberglass cloth sample by positioning the spraybottle ˜4-6 inches from the surface of the fiberglass cloth such thatthe bottle was parallel to the surface of the fiberglass cloth and thenozzle directly faced the fiberglass cloth. The solution was sprayed bydepressing the nozzle continuously for ˜10-12 seconds. The spray bottlewas depressurized after each use and re-pressurized as described abovewhen needed again to ensure equal pressure for delivery of the sprayduring each deposition cycle.

In general, the multilayer films were fabricated on the fiberglass clothsample by spraying the appropriate polyelectrolyte solution or filteredStock OPH solution onto the cloth for 10-12 seconds, allowing the sampleto stand undisturbed for 2 minutes, and spraying the sample 10-12seconds with the appropriate rinse solution (i.e., water or BTP buffer).The assembly comprising the fiberglass cloth sample secured to the first(bottomless) cup by the modified cup lid was then removed from thesecond solution catch basin cup and gently shaken to remove excessliquid from the sample cloth. Said assembly was placed back into saidsecond cup and the process repeated using the appropriate solution todeposit the next layer of polyelectrolyte on OPH enzyme. In order tofabricate the multilayer film bearing a single OPH enzyme layer, thecloth was first sprayed with the BPEI-w solution and then sprayed withwater for rinsing. The second cycle deposited the enzyme by sprayingwith the filtered stock enzyme solution, followed by spraying with theBTP buffer solution for rinsing. The final polyelectrolyte-capping layerwas then applied in the third spraying cycle. BPEI-b solution wassprayed onto the sample cloth, followed by spraying the BTP buffersolution for rinsing.

After this final rinse with BTP buffer, the assembly comprising thefiberglass cloth secured to the first (bottomless) cup by the modifiedplastic lid was removed from the second cup. The modified lid securingthe treated fiberglass sample to the first (bottomless) cup was removedto free the cloth. The area of the cloth exposed directly to the spraysduring treatment was cut from the remaining cloth using scissors. Thisportion of treated cloth was placed with the sprayed side facing up on alint-free clean room paper towel for ˜5 minutes to enhance drying of thesample. The treated fiberglass sample cloth was then transferred to thelyophilizer and subjected to the Standard Drying Protocol before beingstored in the refrigerator.

Example 9 Fabrication of Multilayer-Coated Fiberglass Cloth BearingMultiple Layers of OPH Enzyme by Spray Coating

This example extends the method of Example 8 for the fabrication ofmultilayer-coated fiberglass cloths bearing 3 OPH enzyme layers.

A sample of fiberglass cloth bearing a single layer of OPH enzyme wasfabricated by spray coating as described in Example 8. However,following binding of the capping BPEI-b layer and subsequent rinse inthe BTP solution, treatment was continued by spraying 10-12 seconds withPSS-w solution. The sample was allowed to stand for 2 minutes and thensprayed 10-12 seconds using rinse water. The assembly comprising thecloth sample secured to the first (bottomless) cup using the modifiedlid was removed from the second cup and shaken to dislodge excessliquid. The assembly was replaced into the second cup and the spraytreatment sequence of Example 8 was repeated to apply the next BPEI-wlayer, the second OPH layer, and the next BPEI-b layer. Following sprayapplication of a second PSS-w layer and an aqueous rinse, the treatmentsequence of Example 8 was again performed to apply the third BPEI-w,OPH, and BPEI-b layers. The multilayer sequence of treated sample clothis FG/(BPEI/OPH/BPEI/PSS)₂/BPEI/OPH/BPEI. In general, a multilayercontaining “x” layers of OPH can be prepared through repetition of thespray treatment sequence of Example 8 “x” times, using a PSS layer as aseparation polyelectrolyte layer between each repetition of thetreatment sequence of Example 8.

Example 10 Fabrication of Multilayer-coated Fiberglass Cloth Bearing aSingle Layer of OPH Enzyme by Spin Coating

This example illustrates the fabrication of a multilayer-coatedfiberglass cloth bearing a single layer of OPH enzyme using a spincoating method.

A ˜3.50 inch diameter piece of fiberglass cloth was cleaned using theStandard Cleaning Process and stretched taut over a 3 inch diametersilicon wafer. The fiberglass cloth was secured to the silicon waferusing Mini Binder Clips (Charles Leonard Inc., Catalog No. 50001, 9/16inch size, ¼ inch capacity) of the type used in place of staples tosecure stacks of papers. The clips were mounted uniformly (equidistant)around the circumference of the wafer. The wafer bearing the securedcloth was placed centered on the 2-inch diameter wafer chuck of a SCSspin coater (Model P6204, Specialty Coating Systems Inc., Indianapolis,Ind.). Vacuum was established to the chuck and the spin coater wasactivated. Test runs were made by applying ˜6-8 mL of water to thesample cloth and spinning the sample at various speeds for varioustimes. Use of spinning speeds in excess of ˜1600 rpm for 20-30 secondsreadily removed the water from the sample, but also occasionallyresulted in separation of the wafer from the chuck. It was eventuallydetermined that spinning speeds of 1000 rpm for 20 seconds weresufficient to remove excess liquid from the sample cloth withoutseparation of the wafer from the sample chuck. Consequently, theseconditions were used during the treatment of the sample cloth withvarious polyelectrolyte, enzyme, and rinse solutions as described below.

In general, coating of the fiberglass sample cloth was accomplished bypuddling ˜4-6 mL of the appropriate polyelectrolyte, enzyme, or rinsesolution (i.e., water or BTP buffer) onto the sample cloth, allowing thesolution to stand on the cloth for a short time (i.e., 2 minutes forpolyelectrolyte or Stock OPH solutions and 30 seconds for rinsesolutions), and removing the excess solution by activating the spincoater for 20 seconds at 1000 rpm. The process was repeated until alldepositions and rinses were completed. For the fabrication of amultilayer-coated fiberglass cloth bearing a single OPH enzyme layer,the fiberglass cloth was first treated with BPEI-w solution, spun dry,rinsed with water, and spun dry again. Stock OPH enzyme solution wasnext applied to the fiberglass cloth, which was then spun dry, rinsedwith BTP buffer solution, and spun dry again. Finally, the cloth wastreated with the BPEI-b solution, spun dry, rinsed with BTP buffersolution, and spun dry again. The wafer was removed from the spin coaterchuck and the Mini Binder Clamps were released to remove the treatedfiberglass cloth. The edges of the cloth, which had been covered by theMini Binder Clip or overhung the edge of the silicon wafer during thetreatment process, were trimmed away with scissors and discarded. Theremainder of the treated fiberglass sample cloth was subjected to theStandard Drying Protocol and stored in the refrigerator. The multilayerfilm deposited onto the fiberglass sample cloth has the structureFG/BPEI/OPH/BPEI.

Example 11 MPT Hydrolysis Activity of Multilayer-coated Fiberglass ClothContaining a Single OPH Enzyme Layer Prepared by Spray Coating

This example demonstrates that fiberglass cloth spray coated with amultilayer polyelectrolyte film encapsulating a single layer of OPHenzyme is capable of hydrolyzing MPT in solution to PNP.

The experiment of Example 2 was repeated using a FG/BPEI control clothand a piece of the spray coated FG/BPEI/OPH/BPEI cloth (weight ˜115 mg)prepared in Example 8. The Standard Enzyme Activity Assay indicated thatonly ˜0.8 μM PNP was produced in the MPT solution exposed to the FG/BPEIcontrol cloth, consistent with negligible MPT hydrolysis. In contrast,˜5.7 μM PNP was detected in the MPT solution treated using theFG/BPEI/OPH/BPEI sample cloth, demonstrating the retained activity ofthe encapsulated OPH enzyme towards MPT hydrolysis in this sample.

Example 12 MPT Hydrolysis Activity of Multilayer-Coated Fiberglass ClothContaining Multiple OPH Enzyme Layers Prepared by Spray Coating

This example demonstrates that fiberglass cloth spray coated with amultilayer polyelectrolyte film encapsulating 3 layers of OPH enzyme iscapable of hydrolyzing MPT in solution to PNP.

The experiment of Example 2 was repeated using a FG/BPEI control clothand a piece of the spray coated FG/(BPEI/OPH/BPEI/PSS)₂/BPEI/OPH/BPEIcloth (weight ˜99 mg) prepared in Example 9. The Standard EnzymeActivity Assay indicated that only ˜0.7 μM PNP was produced in the MPTsolution exposed to the FG/BPEI control cloth, consistent withnegligible MPT hydrolysis. In contrast, ˜6.1 μM PNP was detected in theMPT solution treated using the FG/(BPEI/OPH/BPEI/PSS)₂/BPEI/OPH/BPEIsample cloth, demonstrating the retained activity of the encapsulatedOPH enzyme towards MPT hydrolysis in this sample.

Example 13 MPT Hydrolysis Activity of Multilayercoated Fiberglass ClothContaining a Single OPH Enzyme Layer Prepared by Spin Coating

This example demonstrates that fiberglass cloth spin coated with amultilayer polyelectrolyte film encapsulating a single layer of OPHenzyme is capable of hydrolyzing MPT in solution to PNP.

The experiment of Example 2 was repeated using a FG/BPEI control clothand a piece of the spin coated FG/BPEI/OPH/BPEI cloth (weight ˜65 mg)prepared in Example 10. The Standard Enzyme Activity Assay indicatedthat only ˜0.7 μM PNP was produced in the MPT solution exposed to theFG/BPEI control cloth, consistent with negligible MPT hydrolysis. Incontrast, 55.6 μg PNP (4 μM PNP/80 mg silica cloth) was detected in theMPT solution treated using the FG/BPEI/OPH/BPEI sample cloth,demonstrating the retained activity of the encapsulated OPH enzymetowards MPT hydrolysis in this sample.

Example 14 Fabrication of Multilayercoated Cotton Cloth Bearing a SingleLayer of OPH Enzyme by Dip Coating

This example demonstrates the preparation of a polyelectrolyte-enzymemultilayer containing a single layer of the OPH enzyme on a cotton clothsample by the dip coating method.

The experiment described in Example 1 was repeated using a single cottoncloth (Hanes, Inc.), rather than the fiberglass cloth, as the substratesample. A multilayer film of structure CC/BPEI/OPH/BPEI, where theabbreviation CC refers to the cotton cloth substrate, was deposited ontothe cotton cloth. In this case, because an untreated cotton cloth servesas a suitable control, no control sample of structure CC/BPEI analogousto the FG/BPEI of Example 1 was prepared.

Example 15 MPT Hydrolysis Activity of Multilayercoated Cotton ClothContaining a Single OPH Enzyme Layer Prepared by Dip, Coating

This example shows the activity of the dip coated multilayer-coatedcotton cloth of Example 14 having a single OPH enzyme layer towards thehydrolysis of MPT to PNP.

The experiment described in Example 2 was repeated using a piece of theHanes cotton cloth as a control and the CC/BPEI/OPH/BPEI sample (weight˜83 mg) prepared in Example 14. The Standard Enzyme Activity Assayindicated that 1.1 μM was produced in the MPT solution exposed to thecontrol cotton cloth. In contrast, ˜86.5 μM PNP was produced in the MPTsolution exposed to the CC/BPEI/OPH/BPEI sample, consistent witheffective hydrolysis of MPT.

Example 16 Fabrication of Multilayercoated Cotton Cloth Bearing a SingleLayer of OPH Enzyme by Spray Coating

This example describes the fabrication of a multilayer coatingcontaining one layer of OPH enzyme on a cotton cloth using a spraycoating method.

The experiment described in Example 8 was repeated using a single cottoncloth (Hanes, Inc.), rather than the fiberglass cloth, as the substratesample. A multilayer film of structure CC/BPEI/OPH/BPEI was depositedonto the cotton cloth. In this case, because an untreated cotton clothserves as a suitable control, no control sample of structure CC/BPEIanalogous to the FG/BPEI of Example 1 was prepared.

Example 17 MPT Hydrolysis Activity of Multilayercoated Cotton ClothContaining a Single OPH Enzyme Layer Prepared by Spray Coating

This example shows the activity of the spray coated multilayer-coatedcotton cloth of Example 16 having a single OPH enzyme layer towards thehydrolysis of MPT to PNP.

The experiment described in Example 2 was repeated using a piece of theHanes cotton cloth (89 mg) as a control and the CC/BPEI/OPH/BPEI sample(787 mg) prepared in Example 16. The Standard Enzyme Activity Assayindicated that 1.1 μM PNP was produced in the MPT solution exposed tothe control cotton cloth. In contrast, ˜97.1 μM PNP was produced in theMPT solution exposed to the CC/BPEI/OPH/BPEI sample, consistent witheffective hydrolysis of MPT.

Example 18 Fabrication of Multilayercoated Cotton Cloth Bearing a SingleLayer of OPH Enzyme by Spin Coating

This example demonstrates the preparation of a polyelectrolyte-enzymemultilayer containing a single layer of the OPH enzyme on a cotton clothsample by the spin coating method.

The experiment described in Example 10 was repeated using a singlecotton cloth (Hanes, Inc.), rather than the fiberglass cloth, as thesubstrate sample. A multilayer film of structure CC/BPEI/OPH/BPEI wasdeposited onto the cotton cloth. In this case, because an untreatedcotton cloth serves as a suitable control, no control sample ofstructure CC/BPEI analogous to the FG/BPEI of Example 1 was prepared.

Example 19 MPT Hydrolysis Activity of Multilayercoated Cotton ClothContaining a Single OPH Enzyme Layer Prepared by Spin Coating

This example shows the activity of the dip coated multilayer-coatedcotton cloth of Example 18 having a single OPH enzyme layer towards thehydrolysis of MPT to PNP.

The experiment described in Example 2 was repeated using a piece of theHanes cotton cloth (89 mg) as a control and the CC/BPEI/OPH/BPEI sample(480 mg) prepared in Example 18. The Standard Enzyme Activity Assayindicated that 1.1 μM PNP was produced in the MPT solution exposed tothe control cotton cloth. In contrast, 83 μM PNP was produced in the MPTsolution exposed to the CC/BPEI/OPH/BPEI sample, consistent witheffective hydrolysis of MPT.

Example 20 Fabrication of Polyelectrolyte-OPH Enzyme Multilayer onCotton Thread by Dip Coating

This example demonstrates the preparation of a polyelectrolyte-enzymemultilayer containing a single layer of the OPH enzyme on a cottonthread sample by the dip coating method.

An 8-meter length of 100% cotton thread (Mouliné Spécial DMC 25, DMCS:A., F5579 Paris cedex 12, France) was placed loosely in a beakercontaining a Teflon® stirbar. BPEI-w solution sufficient to cover thethread was added to the beaker and gently stirred for 15 minutes. TheBPEI-w solution was decanted from the beaker and the thread was rinsed 3times by immersion in water. The thread was allowed to stand in thefinal water rinse solution for 2 minutes with stirring. The thread wasthen removed from the beaker and pressed between two lint-free papercleanroom towels to remove excess liquid before being returned to theempty beaker. Stock OPH solution was added to the beaker and the samplewas gently stirred for 15 minutes. The enzyme solution was decanted fromthe beaker and subjected to the Standard Enzyme Activity Assay to verifythe activity of the OPH enzyme. The thread in the beaker was immersed inBTP buffer rinse solution with gentle stirring for 2 minutes. The threadwas then removed, gently pressed between two lint-free cleanroom towelsas described above, and placed in a beaker containing stirred BPEI-bsolution for 15 minutes. The BPEI-b solution was then decanted from thebeaker and replaced with stirred BTP rinse buffer. After 2 minutes, thethread was removed from the beaker and again pressed between twolint-free cleanroom towels to remove excess liquid. The thread was thentransferred to the lyophilizer and subjected to the Standard DryingProtocol. The treated thread was stored in the refrigerator until neededfor experiments. The structure of the treated thread wasCT/BPEI/OPH/BPEI, where the abbreviation CT refers to the cotton thread.

Example 21 MPT Hydrolysis Activity of Multilayercoated Cotton ThreadContaining a Single OPH Enzyme Layer Prepared by Dip Coating

This example shows the activity of the dip coated cotton thread ofExample 20 having the structure CT/BPEI/OPH/BPEI towards the hydrolysisof MPT to PNP.

A 15 inch length of the multilayer-coated cotton thread from Example 20having the structure CT/BPEI/OPH/BPEI was loosely packed into the bottomof a plastic cylindrical 50 mL centrifuge tube (Falcon BD). A 15-inchlength of cotton thread, untreated by polyelectrolyte or OPH enzymesolutions, was similarly placed in a second tube. A third tube was leftempty. A 2 mL aliquot of Stock MPT, which completely covered the cottonthreads, was added to each tube. The tubes were capped and allowed tostand undisturbed for 6 hours at 23±2° C. After 6 hours, a StandardEnzyme Activity Assay was performed for the solution in each tube todetermine the concentration of PNP produced by hydrolysis of the MPT.The [PNP] in the control tube, which did not contain any cotton thread,was 0.6 μM, indicating that the MPT solution was stable in the absenceof the cotton threads. The [PNP] present in the tube containing theuntreated cotton thread was 2.0 μM, consistent with minor hydrolysis ofMPT by chemical residues in the thread. In contrast, 98 μM PNP wasobserved in the tube containing the multilayer-coated cotton threadbearing the single layer of OPH enzyme. These results indicate that OPHenzyme immobilized on the cotton thread is an effective catalyst for thehydrolysis of MPT to PNP in solution.

Example 22 MPT Hydrolysis Activity of Multilayer Woven Cotton ClothBearing a Single OPH Enzyme Layer Prepared by Dip Coating

This example demonstrates that OPH enzyme activity towards hydrolysis ofMPT to PNP in solution is maintained for the cotton threads even afterthey are woven into a fabric.

The multilayer-coated cotton thread from Example 20 having the structureCT/BPEI/OPH/BPEI was woven by hand into a square piece of fabric havingdimensions ˜5.0 cm×˜1.5 cm (weight 803 mg). The sample of woven cottonfabric was placed in a plastic cylindrical 50 mL centrifuge tube (FalconBD) and a 20 mL aliquot of fresh Stock MPT solution was added. A secondfabric was woven with dimensions ˜5.0 cm×˜1.5 cm (weight ˜945 mg) fromcotton thread that had not been treated with polyelectrolyte or OPHsolutions as a control. This was placed in a separate centrifuge tubecontaining a 20 mL aliquot of fresh Stock MPT solution. A third tubecontaining only the 20 mL aliquot of fresh Stock MPT solution (no cottonfabric) was also prepared. Each of the tubes was sealed, mounted to alaboratory rotater (Glascol, Terre Haute, Ind., Catalog No. 099ARD4512), and gently agitated overnight (˜14 hours). A Standard EnzymeActivity Analysis was then performed on each solution. The [PNP]produced in the tube containing no cotton fabric was 1 μM, indicatingthat the MPT solution was stable towards extraneous decomposition duringthe experiment. For the sample containing the untreated control cottonfabric, ˜2 μM PNP was measured consistent with the negligible hydrolysisof MPT by the cotton thread exhibited in Example 21. In contrast, [PNP]˜99 μM was measured for the fabric woven from the CT/BPEI/OPH/BPEIcotton thread, consistent with complete MPT hydrolysis during theexperiment. Consequently, sufficient enzymatic activity of the OPH inthe CT/BPEI/OPH/BPEI cotton thread of Example 21 is maintained duringthe mechanical manipulations (and potential abrasion) of the threadduring the process of weaving the thread into a fabric.

Example 23 Time Dependence of the MPT Hydrolysis Activity of MultilayerWoven Cotton Cloth Bearing a Single OPH Enzyme Layer Prepared by DipCoating

This example illustrates the activity of the multilayer-coated wovencotton fabric bearing a single OPH enzyme layer of Example 22 towardsthe repetitive hydrolysis of MPT in solution as a function of time sincefabrication.

The woven cotton fabric from Example 22 was rinsed 3 times with waterand dried by pressing between two layers of lint-free cleanroom towelsat the conclusion of the experiment described in Example 22. It was thentransferred to another 50 mL centrifuge tube (Falcon BD) containing afresh 20 mL aliquot of Stock MPT. The tube was capped, secured to thelaboratory rotator, and again gently agitated overnight (˜14 hours).Afterwards, the Standard Enzyme Activity Analysis performed on thesolution showed that ˜67 μM PNP had formed. The woven cotton fabric wasremoved from the tube, rinsed three times with water, and pressedbetween two lint-free cleanroom towels to remove excess liquid. It wasthen placed in another centrifuge tube containing a fresh 20 mL aliquotof Stock MPT solution, sealed, and gently agitated as described aboveovernight (˜14 hours). The degree of hydrolysis of the MPT again wasmeasured. The amount of PNP generated after this third use cycle was ˜42μM. Consequently, the cotton fabric woven from the CT/BPEI/OPH/BPEIcotton thread is capable of reuse over a several day period whilemaintaining sufficient catalytic activity towards the hydrolysis of MPTto PNP in solution.

Example 24 Determination of the Relative Binding Efficiencies of MPT andPNP in Polycyclodextrin Resin

This example illustrates the relative binding efficiencies of MPT andPNP in a polycyclodextrin resin.

A poly-β-cyclodextrin resin was prepared by reaction of β-cyclodextrinand 1,6-diisocyanatohexane (DICH) in dimethylformamide (DMF) solution.Specifically, 25 grams (˜22 mmoles) of β-cyclodextrin and 30 grams (178mmoles) of DICH were dissolved in 700 mL of DMF. The solution was heatedwith stirring to 90-95° C. for 10 hours. After cooling to roomtemperature, the DMF solution was poured into 1000 mL of stirred waterto precipitate the poly-β-cyclodextrin (PCD) as a fine, white,free-flowing powder. The precipitate was collected by suctionfiltration, washed thoroughly with water, and subjected to the StandardDrying Protocol. A yield of 46 grams of PCD was obtained form theprocedure. The PCD was used without further purification to determinethe relative binding affinities of MPT and PNP as described below.

A 50 mL aliquot of Stock 100 μM MPT and a 50 mL aliquot of Stock 100 μMPNP were mixed in a flask containing a Teflon® stirbar. A 100 mgquantity of PCD was added and the flask was sealed and the contentsstirred at room temperature (23±2° C.). Every 30 minutes, the stirringwas stopped and the solid PCD was allowed to settle. A sample of thesupernatant was obtained and its absorbance at 275 m was measured. Thesample was then returned to the flask, which was again sealed andstirred until the time to take the next sample. The cycle was repeateduntil successive absorbance measurements differed by less than 0.005units, signifying that equilibrium had been reached. At this point, a600 μL sample of solution was removed and diluted with 900 μL of 1 MNaOH(aq) solution. The absorbance of this solution was read at 405 nm.From these absorbance readings, the concentrations of MPT and PNPremaining in the solution were calculated using the calibration curesprepared for the Standard Enzyme Activity Assay. Equilibriumconcentrations of [MPT] ˜16 μM and [PNP] ˜46 μM were measured in thesolution in this manner. Consequently, the PCD bound ˜34 μM MPT and ˜4μM PNP.

The equilibria occurring in the solution in the presence of the solidPCD are defined in equations (3) and (4) below, where [MPT] and [PNP]are the equilibrium concentrations of MPT and PNP, respectively, insolution. K_(m) and K_(p) are the equilibrium binding constants of MPTand PNP, respectively, by the PCD.MPT+PCD

MPT−PCD K _(m) =[MPT−PCD]/([MPT][PCD])  (3)PNP+PCD

PNP−PCD K _(p) =[PNP−PCD]/([PNP][PCD])  (4)

Combining equations (3) and (4) gives Equation (5).S=K _(m) /K _(p)=([MPT−PCD]/[MPT])×([PNP]/[PNP−PCD])  (5)

The term S in equation (5) represents the preference of the PCD forbinding MPT over PNP. The concentration of MPT bound in the PCD,[MPT−PCD], is simply the difference between the initial concentration ofMPT in solution, [MPT]₀, and the concentration remaining at equilibrium,[MPT], as shown in equation (6).[MPT−PCD]=[MPT] ₀ −[MPT]=50 μM−16 μM=34 μM  (6)

Likewise, the concentration of PNP bound in the PCD, [PNP−PCD], issimply the difference between the initial concentration of PCD insolution, [PCD]₀, and the concentration remaining at equilibrium, [PCD],as shown in equation 7.[PNP−PCD]=[PNP] ₀ −[PNP]=50 μM−46 μM=4 μM  (7)

Substitution of equations (6) and (7) into equation (5) yields equation(8).S=K _(m) /K _(p)=(([MPT] ₀ −/[MPT])/[MPT])×([PNP]/([PNP] ₀ −[PNP]))  (8)

Using equation (8) and the measured concentrations of [PNP]₀=[MPT]₀=50μM, [MPT] ˜16 μM, and [PNP] ˜46 μM, we calculate S ˜24. Therefore, bothMPT and PNP are bound by the PCD resin and binding of MPT is favored bya factor of ˜24 over binding of PNP in the PCD resin.

Example 25 Fabrication of a Multilayercoated Cotton Cloth Functionalizedwith β-Cyclodextrin having a Single OPH Enzyme Layer

This example describes the functionalization of a cotton cloth withβ-cyclodextrin and the use of said functionalized cotton cloth as aplatform for fabrication of a multilayer film bearing a single layer ofOPH enzyme.

A square piece of cotton cloth (Hanes Inc.) of size ˜8 inches×8 incheswas placed in a 1000 mL Erlenmeyer flask equipped with Teflon® stirbar.A solution containing ˜10 grams (˜8.81 mmole) of β-cyclodextrin and ˜13grams (˜77.3 mmole) of DICH in ˜200 mL of N,N-dimethylformamide (DMF)was added to the flask. The contents were then heated at 90-95° C. for 4hours. After the flask cooled to room temperature, thePCD-functionalized cotton cloth (CC-PCD) was removed and washedsuccessively with methanol, dichloromethane, acetone, and then copiouslywith water. The treated cloth was then cured by baking in an oven at120° C. for 2 hours and allowed to cool to room temperature. Theexperiment described in Example 1 was then repeated using thePCD-functionalized cotton cloth to fabricate the multilayer film by dipcoating. The structure of the resulting multilayer film wasCC-PCD/BPEI/OPH/BPEI. A Standard Enzyme Binding Assay indicated that˜1.41 μg OPH were deposited per mg of CC-PCD cloth.

Example 26 Fabrication of a Multilayercoated Cotton Cloth Functionalizedwith an Aminoalkylsiloxane Film having a Single OPH Enzyme Layer

This example describes the functionalization of cotton cloth with aminefunctional groups and the use of said functionalized cotton cloth as aplatform for fabrication of a multilayer film bearing a single layer ofOPH enzyme without the need to use an initial BPEI polymer layer.

A 1 gram (˜4.50 mmole) quantity ofN-[3-(trimethoxysilyl)propyl]ethylenediamine (TMSED) was dissolved in 20mL deionized water contained in a 50 mL plastic centrifuge tube. A pieceof cotton cloth (˜5 cm×5 cm) was placed in the solution and the tube wassealed and mounted on the wheel of a laboratory rotator. The sample wasrotated for 20 min at a speed of ˜60 rpm to thoroughly wet the cloth. Atthis point, 200 μL of concentrated ammonium hydroxide solution was addedto the sample solution and agitation was continued using the rotator for3 hours. The cotton cloth was then removed from the TMSED solution,washed 3 times with deionized water, and cured by baking in an oven at125° C. for 2 hours. After the amine-functionalized cloth (CC-NH₂) hadcooled to room temperature, a piece treated with ninhydrin solutionproduced a red color indicative of functionalization by amino groups.The experiment described in Example 1 was then repeated using the CC-NH₂cloth with the omission of the first step involving deposition of theBPEI-w polyelectrolyte layer to fabricate the multilayer film by dipcoating. The structure of the resulting multilayer film wasCC-NH₂/OPH/BPEI. A Standard Enzyme Binding Assay indicated that ˜1.53 μgOPH were deposited per mg of CC-NH₂ cloth.

Example 27 MPT Hydrolysis Activity of β-Cyclodextrin-functionalizedCotton Cloth and Unfunctionalized Cotton Cloth each Coated by aMultilayer Film Containing a Single OPH Enzyme Layer Prepared by DipCoating

This example demonstrates that a PCD-functionalized cotton cloth dipcoated with a multilayer polyelectrolyte film encapsulating a singlelayer of OPH enzyme from Example 25 is capable of hydrolyzing MPT insolution to PNP at a faster rate than a standard CC/BPEI/OPH/BPEI film.

A CC/BPEI/OPH/BPEI cloth standard was prepared as described in Example14. The Standard Enzyme Binding Assay indicated that ˜1.77 μg OPH/mgcloth were bound. A piece of cotton cloth (CC) not treated by BPEI orOPH was also used. Pieces of CC, CC/BPEI/OPH/BPEI, andCC-PCD/BPEI/OPH/BPEI from Example 25, each of size ˜5.0 cm×2.7 cm andweighing ˜155 mg, ˜158 mg, and ˜191 mg, respectively, were prepared bycutting larger sized cloths. Each sample of cloth was placed in aseparate 50 mL plastic centrifuge tube. A 20 mL aliquot of Stock 100 μMMPT solution was added to each centrifuge tube and the tubes were cappedand agitated using the laboratory rotator. After 10, 30, 60, 120, 180,and 300 minutes, aliquots of solution were withdrawn from each tube andthe [PNP] produced was measured using the Standard Enzyme ActivityAssay. MPT hydrolysis activities for each sample were calculated usingthe initial slope (≡initial velocity) of plots of [PNP] versus time. Forthe CC, the activity was essentially zero (i.e., <0.01×10⁻⁹ M·s⁻¹). Forthe CC/BPEI/OPH/BPEI sample, the initial velocity was ˜1.0×10⁻⁹ M·s⁻¹(≡0.32 min⁻¹). For the CC-PCD/BPEI/OPH/BPEI sample, the initial velocitywas ˜1.8×10⁻⁸M·s⁻¹ (≡5.77 min⁻¹), a rate ˜18 times faster than theCC/BPEI/OPH/BPEI sample.

Example 28 MPT Hydrolysis Activity of an Amino-functionalized CottonCloth Coated by a Multilayer Film Containing a Single OPH Enzyme LayerPrepared by Dip Coating

This example demonstrates that a amino-functionalized cotton cloth dipcoated with a multilayer polyelectrolyte film encapsulating a singlelayer of OPH enzyme from Example 26 is capable of hydrolyzing MPT insolution to PNP at a comparable rate to the standard CC/BPEI/OPH/BPEIfilm.

The experiment of Example 27 was repeated using a sample of theCC-NH₂/OPH/BPEI (size ˜5.0 cm×2.7 cm; weight ˜168 mg) prepared accordingto Example 26. An initial velocity for the hydrolysis of MPT was˜1.3×10⁻⁹ M·s⁻¹ (≡0.44 min⁻¹), comparable to that obtained for theCC/BPEI/OPH/BPEI sample in Example 27.

Example 29 PNP Binding Ability of β-Cyclodextrin-functionalized CottonCloth

This example demonstrates that the CC-PCD sample prepared in Example 25binds PNP.

Pieces of CC, CC-PCD prepared from Example 25, and CC-NH₂ prepared fromExample 26, each of size ˜5.5 cm×1.5 cm and weighing ˜89 mg, ˜107 mg,and ˜104 mg, respectively, were prepared by cutting larger sized cloths.Note that these samples are not coated with the BPEI/OPH/BPEImultilayers. Each sample of cloth was placed in a separate 50 mL plasticcentrifuge tube. A 5 mL aliquot of Stock 100 μM PNP solution was addedto each centrifuge tube and the tubes were allowed to stand overnightfor 16 hours. The cloth samples were removed from each PNP solution andthe [PNP] remaining in solution was measured using the proceduredescribed for the Standard Enzyme Activity Assay. A [PNP]>99 μM wasmeasured for solutions containing the CC and CC-NH₂ samples, indicatingthat essentially no PNP was adsorbed to these materials. In contrast,[PNP] ˜87 μM was measured for the solution containing the CC-PCD sample,indicating that PNP adsorption had occurred. The amount of PNP adsorbedby the CC-PCD sample was ˜3.9×10⁻⁹ moles PNP/cm² CC-PCD (≡6.1×10⁻¹⁰moles PNP/mg CC-PCD).

Example 30 Preparation of a β-Cyclodextrin-functionalizedPolyelectrolyte

This example describes the synthesis of a polyelectrolyte bearingcovalently attached β-cyclodextrin group for use in fabrication ofpolyelectrolyte-enzyme multilayer films on substrates.

A BPEI polyelectrolyte bearing pendant β-cyclodextrin groups covalentlyattached to the polymer amine groups was prepared via slightmodification of the literature methods (G. Crini, G. Torri, M. Guerrini,B. Martel, Y. Lekchiri, M. Morcellet, “LinearCyclodextrin-poly(vinylamine): Synthesis and NMR Characterization,” Eur.Polym. J., 33 (7) 1143-1151 (1997); and A. Ruebner, G. L. Statton, M. R.James, “Synthesis of a linear polymer with pendent γ-cyclodextrins,”Macromol. Chem. Phys., 201, 1185-1188 (2000), the entire contents ofboth are incorporated herein by reference) as follows:Mono-6-Tolylsulfonyl-6-deoxy-β-cyclodextrin (TCD) was first prepared bydropwise addition of a solution of 0.151 g ρ-toluenesulfonyl chloride(0.792 mmol) in 5 ml pyridine to a stirred solution of 1.0 gβ-cyclodextrin (0.88 mmol) in 5 ml pyridine at 5° C. under an atmosphereof dry N₂. Following the addition, the solution was stirred for afurther 48 hours at 5° C. under an atmosphere of dry N₂. Afterevaporation of the solvent, the residue was washed repeatedly with waterand acetone followed by drying under vacuum at 60° C. affording 0.388 g(38%) of product. The tosylated cyclodextrin was analyzed in a solventsystem of butanol-ethanol-ammonium hydroxide-water 5:4:4:3. Spots werevisualized by staining and heating with an anthrone solution (0.1 wt-%anthrone in H₂SO₄ diluted 1:50 with ethanol). A single spot on TLC gavea R_(f=)0.75. ¹H NMR (400 MHz, DMSO-d₆): 7.7-7.4 (AA′BB′ 4 H); 5.74 (m,14 H); 4.78-4.79 (m, 7 H); 3.59-3.21, (m, CD protons), 2.52 (s, 3 H).¹³C NMR (100 MHz CDCl₃) 145.1, 133.0, 130.2, 127.9, 102.3, 81.9, 73.3,72.7, 72.3, 60.2, 21.5. TOFHRMS: calculated for C₄₉H₇₆O₃₇S, 1288; found,1289.

Polyethylenimine with 1.2% pendent β-cyclodextrins (BPEI-CD) was thenprepared by dropwise addition of a solution of 0.300 g TCD (0.232 mmol)in 3 mL DMSO to a stirred solution of 0.300 g polyethylenimine in 7 mLDMSO at 60° C. for 48 hours. The crude product was purified by membranefiltration. The residue was freeze-dried affording 0.355 g (59.0% yieldbased on addition of BPEI). The BPEI-CD polymeric system formed wasanalyzed in a solvent system of butanol-ethanol-ammonium hydroxide-water5:4:4:3. Spots were visualized by staining and heating with an anthronesolution (0.1 wt-% anthrone in H₂SO₄ diluted 1:50 with ethanol). Asingle spot on TLC gave an R_(f=)0.0. ¹H NMR (400 MHz, D₂O): 4.79 (bs, 7H), 3.59-3.2 (m, CD protons), 2.92-2.57 (m, PEI). By adjusting theratios of PEI and CD, polyethylenimine with 2.1% β-cyclodextrin loading(BPEI:CD=0.250 g: 0.500 g; ¹H NMR (400 MHz, D₂O): 4.79 (bs, 7 H),3.59-3.2 (m, CD protons), 2.92-2.57 (m, BPEI)) or 3.6% β-cyclode loading(BPEI:CD=0.250 g: 1.00 g; ¹H NMR (400 MHz, D₂O): 4.79 (bs, 7 H),3.59-3.2 (m, CD protons), 2.92-2.57 (m, BPEI)) were also obtained.

The CD loading on the BPEI-CD polymers was determined using theanthrone-CD sugar interaction. A calibration curve was measured withcyclodextrin in a concentration range of 0.01-4.1 mg CD/mL water. Asolution of 0.1 g anthrone in 100 ml conc. H₂SO₄ was prepared. Samplesolutions were prepared by dissolving 10-20 mg BPEI-CD sample in 100 mLwater. 1 mL of the sample solution and 2.5 ml anthrone solution weretransferred into a tube and heated in a water bath at 60° C. for 10minutes. The solutions were cooled to room temperature with cold waterand an UV-VIS spectrum was recorded immediately. The reference cuvettecontained a blank sample (1 mL water+2.5 mL anthrone solution). Theabsorbance at a wavelength of 625 nm was measured. The cyclodextrincontent of the sample was determined from the calibration curve.

Example 31 Fabrication and MPT Hydrolysis Activity of Cotton ClothCoated with a β-Cyclodextrin-functionalized Polyelectrolyte-OPH EnzymeMultilayer Prepared by Dip Coating

This example demonstrates the ability to coat cotton cloth with aβ-cyclodextrin-functionalized polyelectrolyte-OPH enzyme multilayer anduse said coated cloth for the hydrolysis of MPT in solution.

The experiment of Example 1 was repeated using a fresh piece of cottoncloth as the substrate with the following modification. The BPEIpolyelectrolyte in the BPEI-w and BPEI-b solutions was replaced by thecorresponding BPEI-CD polymer prepared in Example 30. The resulting filmhad the structure CC/BPEI-CD/OPH/BPEI-CD. Separate cloth samples wereprepared using β-cyclodextrin-functionalized BPEI having 2.1% and 3.6%β-cyclodextrin loadings. In each case, the concentration of the BPEI-CDsolution used to treat the cloth was 1.2 mg BPEI-CD/mL aqueous or BTPbuffer solution. For each cloth, MPT hydrolysis activity was tested byplacing a cloth sample (Weight ˜159 mg; Size ˜5.0 cm×2.5 cm) in a 20 mLvolume of Stock 100 μM MPT solution in a centrifuge tube and agitatingusing the laboratory rotor. The Standard Enzyme Activity Assay wasperformed to determine the extent of MPT hydrolysis as a function oftime. For the CC/BPEI-CD/OPH/BPEI-CD sample loaded at the 2.1% levelwith β-cyclodextrin, an initial velocity of 2.6×10⁻⁹ M·s⁻¹ was observedat 23±2° C. After ˜22 hours, the amount of PNP the amount of PNPproduced was ˜76 μM, indicating substantial hydrolysis of the MPT. Forthe CC/BPEI-CD/OPH/BPEI-CD sample loaded at the 3.6% level withβ-cyclodextrin, an initial velocity of 2.8×10⁻⁹ M·s⁻¹ was observed at23±2° C. After ˜22 hours, the amount of PNP produced was ˜82 μM. Acontrol CC/BPEI/OPH/BPEI cloth, prepared using the same OPH solution andat the same time as the CC/BPEI-CD/OPH/BPEI-CD samples, gave an initialvelocity of ˜1.4×10⁻⁹ M·s⁻¹ and produced ˜59 μM PNP after 22 hours.

Example 32 Vapor Phase MPT Hydrolysis Activity of MultilayercoatedCotton Cloth Containing a Single OPH Enzyme Layer Prepared by DipCoating

This example demonstrates that cotton cloth coated with a multilayerpolyelectrolyte film encapsulating a single layer of OPH enzyme iscapable of hydrolyzing MPT vapors to PNP at 40° C.

A piece of cotton cloth coated with a multilayer film (weight ˜152 mg;size ˜5.2 cm×2.5 cm) as described in Example 14 of structureCC/BPEI/OPH/BPEI was attached by a piece of string to the underside of alid for a jar of volume ˜1330 cm³. A Petri dish containing 5.2 mg solidMPT was placed in the jar and a 500 μL drop of water was placed insidethe jar on the floor. The jar was sealed with the lid such that theCC/BPEI/OPH/BPEI sample was suspended ˜2 cm above the MPT in the Petridish inside the jar. The entire assembly was placed in a water bath heldat 40±1° C. and observed. After 6 days, the cloth exhibited anoticeable, but pale, yellow tint. By the seventh day, the cloth wasclearly yellow in color, suggesting that hydrolysis of MPT vapor hadoccurred on or in the multilayer film coating the cloth. Hydrolysis wasconfirmed by removing the cloth from the jar and extracting it twicewith 2 mL portions of methanol. The yellow methanol extracts werecombined and allowed to evaporate to dryness, yielding a yellow residue.The residue was taken up in 3 mL CHES-m buffer solution and analyzedaccording to the procedure of the Standard Enzyme Activity Assay. Fromthe solution absorbances measured at 275 nm and 405 nm, it was shownthat ˜102 μg MPT and ˜10 μg PNP were present, consistent with adsorptionof MPT vapor and its hydrolysis to PNP by the CC/BPEI/OPH/BPEI sample.

The above description is that of a preferred embodiment of theinvention. Various modifications and variations are possible in light ofthe above teachings. It is therefore to be understood that, within thescope of the appended claims, the invention may be practiced otherwisethan as specifically described. Any reference to claim elements in thesingular, e.g. using the articles “a,” “an,” “the,” or “said” is notconstrued as limiting the element to the singular.

1. A catalytic enzyme-modified substrate for providing protectionagainst chemical agents, comprising: (a) a substrate which absorbscharged polymer components; (b) a polyelectrolyte layer deposited ontothe substrate; (c) an enzyme layer deposited onto the polyelectrolytelayer to degrade a chemical agent; (d) a capping layer deposited ontothe enzyme layer; and (e) optionally a base layer deposited onto thecapping layer, a second polyelectrolyte layer deposited onto the baselayer, a second enzyme layer deposited onto the second polyelectrolytelayer, and a second capping layer; wherein the optional layers in (e)may be repeated any number of times.
 2. The catalytic enzyme-modifiedsubstrate of claim 1, wherein said substrate comprises fiberglass,cotton, rayon, nylon, or any combination thereof.
 3. The catalyticenzyme-modified substrate of claim 1, wherein said substrate comprisesunmodified cotton, cotton modified with cyclodextrin, cotton modifiedwith an amine, or any combination thereof.
 4. The catalyticenzyme-modified substrate of claim 1, wherein said substrate comprises amaterial whose surface has been chemically modified to generatefunctional groups that can support adsorption of charged polymercomponents.
 5. The catalytic enzyme-modified substrate of claim 3,wherein said substrate comprises polytetrafluoroethylene (PTFE) that hasbeen oxidized.
 6. The catalytic enzyme-modified substrate of claim 1,wherein said substrate is a thread that is woven into a fabric afterdeposition of the final capping layer.
 7. The catalytic enzyme-modifiedsubstrate of claim 1, wherein said polyelectrolyte layer comprises aβ-cyclodextrin-functionalized polyelectrolyte.
 8. The catalyticenzyme-modified substrate of claim 1, wherein said enzyme layercomprises organophosphorous hydrolase (OPH), organophosphorous acidanhydrolase (OPAA), DFPase, phosphotriesterases (PTE), or anycombination thereof.
 9. The catalytic enzyme-modified substrate of claim1, wherein said polyelectrolyte layer comprises branched or linearpolyethyleneimine (PEI), polyacrylic acid (PAA), polymethacrylic acid(PMA), polystyrene sulfonate (PSS), polydiallyl dimethyl ammoniumchloride (PDDA), polyvinylpyridine (PVP), polyvinyl sulfate (PVS),polyallylamine hydrochloride (PAH), their chemically alteredderivatives, or any combination thereof.
 10. The catalyticenzyme-modified substrate of claim 1, wherein said capping layercomprises a readily polymerizable monomer.
 11. The catalyticenzyme-modified substrate of claim 1, wherein said capping layercomprises polystyrene sulfonate (PSS), branched polyethylenimine (BPEI),1,2-dihydroxypropyl methacrylate (DHPM), 1,2-dihydroxypropyl4-vinylbenzyl ether (DHPVB), N-[3-trimethoxysilyl)propyl]ethylenediamine(TMSED), or any combination thereof.
 12. The catalytic enzyme-modifiedsubstrate of claim 1, wherein said layers are deposited using dipcoating, spin coating, spray coating, or any combination thereof. 13.The catalytic enzyme-modified substrate of claim 1, wherein theoutermost capping layer is a bactericidal layer.
 14. The catalyticenzyme-modified substrate of claim 13, wherein said outmost cappinglayer comprises branched polyethylenimine (BPEI) modified with a hexylgroup and quaternized with methyl bromide.
 15. A catalyticenzyme-modified substrate for providing protection against chemicalagents, comprising: (a) a substrate comprising cotton modified with anamine; (b) an enzyme layer deposited onto the substrate to degrade achemical agent; (c) a capping layer deposited onto the enzyme layer; and(d) optionally a base layer deposited onto the capping layer, a secondenzyme layer deposited onto the base layer, and a second capping layer;wherein the optional layers in (d) may be repeated any number of times.